A central hurdle in developing small interfering RNAs (siRNAs) as therapeutics is the inefficiency of their delivery across the plasma and endosomal membranes to the cytosol, where they interact with ...the RNA interference machinery. With the aim of improving endosomal release, a poorly understood and inefficient process, we studied the uptake and cytosolic release of siRNAs, formulated in lipoplexes or lipid nanoparticles, by live-cell imaging and correlated it with knockdown of a target GFP reporter. siRNA release occurred invariably from maturing endosomes within ~5-15 min of endocytosis. Cytosolic galectins immediately recognized the damaged endosome and targeted it for autophagy. However, inhibiting autophagy did not enhance cytosolic siRNA release. Gene knockdown occurred within a few hours of release and required <2,000 copies of cytosolic siRNAs. The ability to detect cytosolic release of siRNAs and understand how it is regulated will facilitate the development of rational strategies for improving the cytosolic delivery of candidate drugs.
Full text
Available for:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
Conjugation of small interfering RNA (siRNA) to an asialoglycoprotein receptor ligand derived from N-acetylgalactosamine (GalNAc) facilitates targeted delivery of the siRNA to hepatocytes in vitro ...and in vivo. The ligands derived from GalNAc are compatible with solid-phase oligonucleotide synthesis and deprotection conditions, with synthesis yields comparable to those of standard oligonucleotides. Subcutaneous (SC) administration of siRNA–GalNAc conjugates resulted in robust RNAi-mediated gene silencing in liver. Refinement of the siRNA chemistry achieved a 5-fold improvement in efficacy over the parent design in vivo with a median effective dose (ED50) of 1 mg/kg following a single dose. This enabled the SC administration of siRNA–GalNAc conjugates at therapeutically relevant doses and, importantly, at dose volumes of ≤1 mL. Chronic weekly dosing resulted in sustained dose-dependent gene silencing for over 9 months with no adverse effects in rodents. The optimally chemically modified siRNA–GalNAc conjugates are hepatotropic and long-acting and have the potential to treat a wide range of diseases involving liver-expressed genes.
Full text
Available for:
IJS, KILJ, NUK, PNG, UL, UM
Covalent attachment of a synthetic triantennary N-acetylagalactosamine (GalNAc) ligand to chemically modified siRNA has enabled asialoglycoprotein (ASGPR)-mediated targeted delivery of ...therapeutically active siRNAs to hepatocytes in vivo. This approach has become transformative for the delivery of RNAi therapeutics as well as other classes of investigational oligonucleotide therapeutics to the liver. For efficient functional delivery of intact drug into the desired subcellular compartment, however, it is critical that the nucleic acids are stabilized against nucleolytic degradation. Here, we compared two siRNAs of the same sequence but with different modification pattern resulting in different degrees of protection against nuclease activity. In vitro stability studies in different biological matrices show that 5'-exonuclease is the most prevalent nuclease activity in endo-lysosomal compartments and that additional stabilization in the 5'-regions of both siRNA strands significantly enhances the overall metabolic stability of GalNAc-siRNA conjugates. In good agreement with in vitro findings, the enhanced stability translated into substantially improved liver exposure, gene silencing efficacy and duration of effect in mice. Follow-up studies with a second set of conjugates targeting a different transcript confirmed the previous results, provided additional insights into kinetics of RISC loading and demonstrated excellent translation to non-human primates.
Hemophilia A and B are inherited bleeding disorders characterized by deficiencies in procoagulant factor VIII (FVIII) or factor IX (FIX), respectively. There remains a substantial unmet medical need ...in hemophilia, especially in patients with inhibitory antibodies against replacement factor therapy, for novel and improved therapeutic agents that can be used prophylactically to provide effective hemostasis. Guided by reports suggesting that co-inheritance of prothrombotic mutations may ameliorate the clinical phenotype in hemophilia, we developed an RNA interference (RNAi) therapeutic (ALN-AT3) targeting antithrombin (AT) as a means to promote hemostasis in hemophilia. When administered subcutaneously, ALN-AT3 showed potent, dose-dependent, and durable reduction of AT levels in wild-type mice, mice with hemophilia A, and nonhuman primates (NHPs). In NHPs, a 50% reduction in AT levels was achieved with weekly dosing at approximately 0.125 mg/kg, and a near-complete reduction in AT levels was achieved with weekly dosing at 1.5 mg/kg. Treatment with ALN-AT3 promoted hemostasis in mouse models of hemophilia and led to improved thrombin generation in an NHP model of hemophilia A with anti-factor VIII inhibitors. This investigational compound is currently in phase 1 clinical testing in subjects with hemophilia A or B.
Full text
Available for:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SBMB, UILJ, UKNU, UL, UM, UPUK
The way it is: Incorporation of 2′‐deoxy‐2′‐fluororibonucleotides (2′‐F RNA) into RNA oligomers leads to a marked increase in duplex stability. Using NMR spectroscopy (see picture) with native (red ...circles) and 2′‐F modified (black squares) RNA duplexes, and UV melting and osmotic stress experiments the higher stability of 2′‐F RNA relative to RNA is shown to be the result of both increased Watson–Crick H‐bonding strength and favorable base stacking.
Full text
Available for:
BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
We recently demonstrated that siRNAs conjugated to triantennary N‐acetylgalactosamine (GalNAc) induce robust RNAi‐mediated gene silencing in the liver, owing to uptake mediated by the ...asialoglycoprotein receptor (ASGPR). Novel monovalent GalNAc units, based on a non‐nucleosidic linker, were developed to yield simplified trivalent GalNAc‐conjugated oligonucleotides under solid‐phase synthesis conditions. Synthesis of oligonucleotide conjugates using monovalent GalNAc building blocks required fewer synthetic steps compared to the previously optimized triantennary GalNAc construct. The redesigned trivalent GalNAc ligand maintained optimal valency, spatial orientation, and distance between the sugar moieties for proper recognition by ASGPR. siRNA conjugates were synthesized by sequential covalent attachment of the trivalent GalNAc to the 3′‐end of the sense strand and resulted in a conjugate with in vitro and in vivo potency similar to that of the parent trivalent GalNAc conjugate design.
Sugar goes straight to the liver: An siRNA conjugate bearing three N‐acetylgalactosamine moieties, each sequentially attached by a non‐nucleosidic linker, was recognized by the asialoglycoprotein receptor with high affinity. The resulting liver‐specific delivery of the conjugate for robust RNAi‐mediated gene silencing in vivo shows potential for delivery of oligonucleotide therapeutics into hepatocytes.
Full text
Available for:
FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Asialoglycoprotein receptor (ASGPR) mediated delivery of triantennary N-acetylgalactosamine (GalNAc) conjugated short interfering RNAs (siRNAs) to hepatocytes is a promising paradigm for RNAi ...therapeutics. Robust and durable gene silencing upon subcutaneous administration at therapeutically acceptable dose levels resulted in the advancement of GalNAc-conjugated oligonucleotide-based drugs into preclinical and clinical developments. To systematically evaluate the effect of display and positioning of the GalNAc moiety within the siRNA duplex on ASGPR binding and RNAi activity, nucleotides carrying monovalent GalNAc were designed. Evaluation of clustered and dispersed incorporation of GalNAc units to the sense (S) strand indicated that sugar proximity is critical for ASGPR recognition, and location of the clustered ligand impacts the intrinsic potency of the siRNA. An array of nucleosidic GalNAc monomers resembling a trivalent ligand at or near the 3′ end of the S strand retained in vitro and in vivo siRNA activity, similar to the parent conjugate design. This work demonstrates the utility of simple, nucleotide-based, cost-effective siRNA–GalNAc conjugation strategies.
Full text
Available for:
IJS, KILJ, NUK, PNG, UL, UM, UPUK
The hepatocyte-specific asialoglycoprotein receptor (ASGPR) is an ideal candidate for targeted drug delivery to the liver due to its high capacity for substrate clearance from circulation together ...with its well-conserved expression and function across species. The development of GalNAc-siRNA conjugates, in which a synthetic triantennary N-acetylgalactosamine-based ligand is conjugated to chemically modified siRNA, has enabled efficient, ASGPR-mediated delivery to hepatocytes. To investigate the potential impact of variations in receptor expression on the efficiency of GalNAc-siRNA conjugate delivery, we evaluated the pharmacokinetics and pharmacodynamics of GalNAc-siRNA conjugates in multiple pre-clinical models with reduced receptor expression. Despite greater than 50% reduction in ASGPR levels, GalNAc conjugate activity was retained, suggesting that the remaining receptor capacity was sufficient to mediate efficient uptake of potent GalNAc-siRNAs at pharmacologically relevant dose levels. Collectively, our data support a broad application of the GalNAc-siRNA technology for hepatic targeting, including disease states where ASGPR expression may be reduced.
Willoughby and colleagues show that GalNAc-siRNA conjugate uptake is specifically mediated through the hepatic expressed asialoglycoprotein receptor and that potent conjugates are capable of robust gene silencing in reduced receptor settings. These data combined support the therapeutic potential in disease(s) where receptor levels may be reduced due to liver injury.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Maintenance of normal endothelial function is critical to various aspects of blood vessel function, but its regulation is poorly understood. In this study, we show that disruption of baseline ...fibroblast growth factor (FGF) signaling to the endothelium leads to a dramatic reduction in let-7 miRNA levels that, in turn, increases expression of transforming growth factor (TGF)-β ligands and receptors and activation of TGF-β signaling, leading to endothelial-to-mesenchymal transition (Endo-MT). We also find that Endo-MT is an important driver of neointima formation in a murine transplant arteriopathy model and in rejection of human transplant lesions. The decline in endothelial FGF signaling input is due to the appearance of an FGF resistance state that is characterized by inflammation-dependent reduction in expression and activation of key components of the FGF signaling cascade. These results establish FGF signaling as a critical factor in maintenance of endothelial homeostasis and point to an unexpected role of Endo-MT in vascular pathology.
Display omitted
► FGF controls let-7 miRNA expression in endothelial cells ► A reduction in let-7 expression leads to endothelial-to-mesenchymal transition (Endo-MT) ► Endo-MT is an important contributor to human disease pathology
Maintenance of normal endothelial homeostasis is critical to blood vessel function. In this study, Simon and colleagues demonstrate that disruption of fibroblast growth factor (FGF) signaling input to the endothelium reduces let-7 miRNA levels and activates TGF-β signaling, leading to endothelial-to-mesenchymal transition (Endo-MT). Endo-MT is an important driver of neointima formation in a number of animal models and in rejection of human transplant lesions. These results establish FGF signaling as a critical factor in maintenance of vascular homeostasis and point to an unexpected role of Endo-MT in vascular pathology.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
PDF
